Phosphorylation of MLL by ATR is Required for Execution of Mammalian S Phase Checkpoint

Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Nature (Impact Factor: 41.46). 09/2010; 467(7313):343-6. DOI: 10.1038/nature09350
Source: PubMed


Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.

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    • "Consistently, ChIP analysis of H3K4me3 at the B-globin replication origin in Jurkat cells shows that H3K4me3 does not correlate with CDC45 enrichment [20]. Notably, in cells expressing a catalytic dead mutant of MLL, the H3K4me3 methyltransferase, CDC45 does accumulate at this origin [20]. Thus, H3K4me3 could prevent the binding of CDC45. "
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    ABSTRACT: In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin is critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process. This article is part of a Special Issue entitled: Methylation Multifaceted Modification - looking at transcription and beyond, edited by Dr. Johnathan Whetstine.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 12/2014; DOI:10.1016/j.bbagrm.2014.03.009 · 6.33 Impact Factor
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    • "Previous studies including ours have indicated that MLL participates in the progression of S phase (22–24,26,30). MLL complexes with Host cell factor-1 (HCF-1) to interact with E2F1 and activate S-phase genes (23). "
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    ABSTRACT: MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our analysis reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL–WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku458 · 9.11 Impact Factor
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    • "Antibodies were detected using the enhanced chemiluminescence method (Western Lightning, PerkinElmer). Immunoblot signals were acquired with the LAS-3000 Imaging system (FujiFilm) and were analyzed with ImageGauge software (FujiFilm) as previously described (Liu et al., 2010). "
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    ABSTRACT: Chromosomal translocations disrupting MLL generate MLL-fusion proteins that induce aggressive leukemias. Unexpectedly, MLL-fusion proteins are rarely observed at high levels, suggesting excessive MLL-fusions may be incompatible with a malignant phenotype. Here, we used clinical proteasome inhibitors, bortezomib and carfilzomib, to reduce the turnover of endogenous MLL-fusions and discovered that accumulated MLL-fusions induce latent, context-dependent tumor suppression programs. Specifically, in MLL pro-B lymphoid, but not myeloid, leukemias, proteasome inhibition triggers apoptosis and cell cycle arrest involving activation cleavage of BID by caspase-8 and upregulation of p27, respectively. Furthermore, proteasome inhibition conferred preliminary benefit to patients with MLL-AF4 leukemia. Hence, feasible strategies to treat cancer-type and oncogene-specific cancers can be improvised through harnessing inherent tumor suppression properties of individual oncogenic fusions.
    Cancer cell 04/2014; 25(4):530-42. DOI:10.1016/j.ccr.2014.03.008 · 23.52 Impact Factor
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